Chemokine receptors differentially expressed by race category and molecular subtype in the breast cancer TCGA cohort

Racial disparities in mortality due to metastasis remain significant among breast cancer patients. Chemokine receptors contribute to breast tumors and metastatic outcome. We explored for significant differences in chemokine receptor expression in breast tumors from Black, Asian, and White patients in The Cancer Genome Atlas. We show that despite sharing the same molecular subtype, expression of the chemokine receptors ACKR1, CCR3, CCR6, CCRL1, CCRL2, CXCR1, CXCR2, CXCR4, CXCR6, and CXC3CR1 was significantly different depending on racial group. For patients with triple negative breast cancer, CCR3 was higher in Black versus White and CCRL2 was higher in Asian versus White. In luminal A tumors, ACKR1 was lower in Asian versus White, CCR3 was higher in Black versus White, and CCR6 and CXC3CR1 were lower in Black versus White. In luminal B tumors, CCRL2 was lower in Black versus White, CXCR1 and CXC3CR1 were lower in Asian versus White, and CXCR2 was lower in Black and Asian versus White. In HER2 enriched tumors, CCR3 was higher in Black versus White and CXCR4 lower in Asian versus White. CCR3, CCR6, and CXCR6 associated with worse patient survival. These findings can inform improved treatment strategies to decrease racial disparities in breast cancer burden.

). The top differentially expressed genes in tumors between Black and White patients after adjusting for covariate effects, were RPL29P2 and CRYBB2. The top differentially expressed genes in tumors between Asian and White patients, after adjusting for covariates, were CHGB and LEP (supplementary Tables 2  and 4). Table 1 shows ACKR1/DARC was the only common chemokine receptor DEG between Black versus White and Asian versus White patient tumor samples. CCR3 and CX3CR1 were differentially expressed between Black versus White, and CCR6, CCRL2, and CXCR4 between Asian versus White breast tumors.
Differences between normal and tumor tissues have been well described 31,32 . Significant differences in gene expression in normal versus tumor samples within the breast cancer TCGA cohort have been reported before 31 . However, when we queried for differentially expressed genes in the limited available normal adjacent versus tumor tissues (Supplementary-Tables 5 and 6), we identified only the chemokine receptors CXCR2, CXCR6, and CX3CR1 to be commonly differentially expressed in normal versus tumor samples for both Black and White patients. We were unable to do normal versus tumor analysis for the Asian group due to the lack of normal tissue samples for Asian patients in the breast cancer TCGA.
Gene expression analysis. We plotted the tumor gene expression data to illustrate how the chemokine receptor genes are expressed across race and tumor molecular subtype. Figure 2 shows ACKR1, CCR3, CCR6, CCRL2, CXCR2, and CX3CR1 expression is significantly different based on race category (White, Black or Asian), using the breast cancer TCGA data for tumor samples.
Since breast tumor molecular subtype (Luminal A or B, HER2 enriched, and triple negative), is a well-established determinant of differences in patient outcome 33 , we analyzed receptor expression within molecular subtypes across racial categories. Figure 3 shows ACKR1, CCR3, CCR6, ACKR4/CCRL1, CCRL2, CXCR1, CXCR2, CXCR4, CXCR6, and CX3CR1 expression is significantly different based on breast cancer molecular subtypes. Figure 4 shows that for patients with triple negative breast cancer, CCR3 is significantly higher in Black versus White patients while CCRL2 is higher in Asian versus White patients. Figure 5 shows that for patients with luminal A breast cancer, expression of ACKR1 is lower in Asian than White, CCR3 is higher in Black versus White, and CCR6 and CXC3CR1 are lower in Black versus White.    www.nature.com/scientificreports/ sis between Black, White and Asian patient normal and tumor samples. We used overall survival (OS) for breast cancer tumors in TCGA and in the GEO repository to determine if receptor expression correlated with survival. Figure 8 shows Kaplan-Meier plots for chemokine receptors ACKR1, CXCR6, CCR6, and CX3CR1, which were the only receptors that significantly correlated with the overall survival of patients with breast cancer. We also assessed differences in chemokine receptors expression by race. With a p value of 0.05, higher expression of CXCR6 correlated with better overall survival for Black patients only. No significant receptor correlations with survival were observed for White or Asian breast cancer patients in the TCGA breast cancer cohort. We also looked at the Relapse-free survival rate from patients that survived with no symptoms of cancer, and found no significant differences in survival rate based on these receptor genes. Next, we assessed differences in chemokine receptors expression by molecular subtypes. Figure 9 shows the chemokine receptors that significantly correlated with breast cancer overall survival in basal tumors. Figure 10 shows the receptors that significantly correlated with overall survival rate for breast cancer tumor samples of HER2 enriched, luminal A, and luminal B subtypes.
Chemokine receptor specific findings. ACKR1/DARC . Atypical Chemokine Receptor 1 or Duffy Antigen Receptor for Chemokines (ACKR1/DARC) is an internalizing receptor or chemokine-scavenging/decoy receptor whose ligand binding results in chemokine sequestration, degradation, or transcytosis because of its inability to signal via classic G-protein-mediated pathways. ACKR1 can regulate chemokine bioavailability and leukocyte recruitment when expressed on endothelial cells. It is also expressed by erythrocytes where it serves as a blood reservoir of cognate chemokines but also as a chemokine sink, buffering potential surges in plasma chemokine levels. ACKR1 can inhibit breast cancer growth and progression by sequestration of angiogenic chemokines and subsequent inhibition of tumor neovascularity 34 . DEG analysis showed that ACKR1 is significantly downregulated in Black versus White patients (Table 1), consistent with a recent study showing ACKR1 tumor expression in breast cancer is lower in African American patient's breast cancer tumors than in European American patient tumors 22 . RNA count expression plotting for the TCGA breast cancer cohort only confirmed a significant decrease in ACKR1 expression in Asian compared to White and to Black patients (Fig. 2). In addition, there was no significant difference within the triple negative molecular subtype across the different racial categories (Fig. 4). All our different types of analyses did however, support that ACKR1 is significantly downregulated in Asian versus White and specifically in luminal A patient samples. ACKR1 expression varies significantly based on molecular subtype, with higher expression in luminal A than luminal B, HER2 enriched and triple negative breast tumors. ACKR1 associated with better overall survival (Fig. 8A).
CCR3. The CC Motif Chemokine Receptor 3, CCR3, is expressed in eosinophils, basophils, T, NKT, and airway epithelial cells. It may contribute to accumulation and activation of eosinophils and other inflammatory cells 35 . CCR3 has been associated with improved relapse-free survival in breast cancer with high expression of CCR3 in luminal-like rather than triple negative or HER2 enriched tumors 36 . Our results for the breast TCGA cohort show that CCR3 is higher in the triple negative subtype than in luminal A and B (Fig. 3). In addition, we found CCR3 was upregulated in luminal A, HER2 enriched, and triple negative tumors of only Black versus White patients.
CCR6. The CC Motif Chemokine Receptor 6, CCR6, is expressed by immature dendritic cells and memory T cells. The ligand of this receptor is macrophage inflammatory protein 3 alpha (MIP-3 alpha) 35 . CCR6 is highly expressed in pro-tumorigenic macrophages within the mammary gland microenvironment, promoting breast cancer tumors 37 . CCR6 was differentially expressed, being down regulated in Asian versus White (Table 1) and in Black versus White patients (by expression count data) (Fig. 2). High expression of CCR6 significantly associated with worse overall breast cancer patient survival (Fig. 8D), but correlated with better survival in basal and HER enriched patients (Fig. 9C, 10C). We found CCR6 is higher in luminal A and in triple negative than in luminal B breast cancers. We also found higher CCR6 expression in Black versus White patients with luminal A molecular subtype breast tumors.
CCRL1. The atypical chemokine receptor 4/CC-chemokine receptor-like 1, ACKR4/CCRL1, or CCX-CKR, is expressed by cancer cells, thymic epithelial cells, bronchial cells, and keratinocytes. ACKR4/CCRL1 down regulation correlates with worse outcome in breast cancer. CCRL1 is a negative regulator of growth and metastasis in breast cancer by sequestering chemokines and inhibiting intratumoral neovascularity 38,39 . DEG analysis shows CCRL1 was downregulated across the Black versus White patient samples in the TCGA breast cohort (Table 1). Figure 3 shows CCRL1 is significantly lower in luminal B compared to triple negative breast cancer samples. High ACKR4/CCRL1 expression correlated with worse survival in patients with triple negative or basal tumors (Fig. 9D).
CCRL2. The CC Motif Chemokine Receptor Like 2, CCRL2, is expressed at high levels in endothelial cells, activated macrophages, and myeloid derived leukocytes 35,40,41 . CCRL2 is a receptor for CCL19 and the chemokine/ adipokine chemerin (RARRES2). CCRL2 modulates chemokine-triggered immune responses by capturing and internalizing CCL19 or presenting RARRES2 ligand to the receptor CMKLR1. CCRL2 on activated endothelial cells acts in concert with CMKLR1 to coordinate chemerin-dependent leukocyte adhesion in vitro and recruitment in vivo 41 . CCRL2 expressing adipose tissue progenitor cells cooperate to induce epithelial-to-mesenchymal transition (EMT) gene expression in luminal breast cancer cells to enhance tumor progression and metastatic dissemination 42 . This may be important in the obesity-driven inflammatory microenvironment known to correlate with tumor development and progression in post-menopausal women 42  www.nature.com/scientificreports/ A, B, and triple negative breast tumors. CCRL2 was higher in Asian than White patients with triple negative breast cancer. CCRL2 was also higher in White than in Black patients with luminal B breast tumors. High CCRL2 correlated with better survival in triple negative or basal tumors (Fig. 9E).
CXCR1 and CXCR2. Chemokine receptors CXCR1 and CXCR2 are G-protein-coupled receptors whose biological effects are mediated by the inflammatory chemokine CXCL8 43 . While not abundant in cancer cells, CXCR1 was identified on breast cancer stem-like cells (CSCs) where it plays a major role in regulating the breast CSCs and surrounding cancer cell survival via Fas-ligand mediated pathways 44,45 . Similarly, CXCR2 expression is not consistently found to be highly expressed in cancer cells within breast tumors, but rather this receptor is most highly expressed in the stromal cells of breast tumors, particularly neutrophils. High levels of CXCR2 are also associated with higher infiltration of T and B lymphocytes in breast tumors 46 . We showed that both CXCR1 and CXCR2 are downregulated in Black versus White patients based on RNA counts (Fig. 2). DEG analysis indicates downregulation of both CXCR1 and CXCR2 in the Asian versus White breast cohort (Table 1). Only CXCR2 was downregulated for both Black versus White and Asian versus White patients in the TCGA cohort (Table 1). However, DEG results for tumor only samples did not indicate significant differences for these two receptors based on race category (Table 1). Molecular subtype analysis revealed CXCR1 is significantly lower in triple negative than in luminal A, B and HER2 enriched breast cancers. CXCR2 is significantly lower in luminal B and HER2 enriched than in luminal A tumors (Fig. 3). When analyzing race category within the luminal B subtype, we found that CXCR1 was lower in Asian versus White breast tumors. Similarly, within the luminal B subtype, CXCR2 expression is lower in Black and Asian compared to White patient breast tumors (Fig. 6). CXCR2 expression correlated with better survival in HER2 enriched tumors (Fig. 10B).
CXCR4. CXCR4 is a G-protein-coupled receptor highly expressed in breast cancer cells. CXCR4 plays a role in the growth and metastasis of breast cancer via its ligand CXCL12/SDF-1, which activates mTOR to promote the epithelial-mesenchymal transition (EMT), and is therefore important in metastasis 47 . CXCR4 inhibitors can impair tumor growth and metastatic dissemination in HER2 enriched breast cancer cells but do not reduce www.nature.com/scientificreports/ tumor growth, and can increase metastatic spread of triple negative breast cancer. CXCR4 inhibitors also reduce myofibroblast content in all breast cancer subtypes, but only decrease angiogenesis in HER2 enriched breast cancer. This is surprising considering that we would expect the more aggressive triple negative breast cancer would harbor a more angiogenic and metastatic microenvironment with increased CXCR4 expression. DEG analysis showed CXCR4 was differentially downregulated only for the Asian versus White breast cohort and samples (Table 1). Molecular subtype analysis showed CXCR4 expression was higher in triple negative samples than in luminal A, B, and HER2 enriched. CXCR4 was also higher in HER2 enriched than in luminal A and B (Fig. 3). We only found a significant difference for Asian patients with HER2 enriched, who have higher CXCR4 expression than White patients with HER2 enriched tumors (Fig. 7). CXCR4 expression correlated with better overall survival of patients with basal tumors (Fig. 9B).
CXCR6. CXCR6 is a G-protein-coupled receptor enriched in inflamed tissue lymphoid cells, and also expressed in some epithelial and nonepithelial cancer cells. CXCR6 mediates tumor promoting inflammation via its ligand CXCL16 by inducing macrophage polarization toward a pro-tumoral phenotype in solid tumors 18 . Reduction of CXCR6 expression in breast cancer mouse models decreases metastasis of those tumors 48 . DEG analysis indicated CXCR6 was upregulated in the Asian versus White TCGA cohort (Table 1). Molecular subtype analysis showed CXCR6 is significantly higher in triple negative and in HER2 enriched than in luminal A and B breast tumors (Fig. 3). Higher expression of CXCR6 correlated with slightly worse overall patient survival (Fig. 8B) while CXCR6 correlated with slightly better survival for Black patients (not shown) and better survival for patients with basal tumors (Fig. 9C). This suggests that CXCR6 is potentially one of the chemokine receptors mediating pro-inflammatory microenvironment in Asian and Black breast tumors.

CX3CR1.
The chemokine receptor CX3CR1 is over-expressed in both primary and metastatic breast tumors. CX3CR1 determines the arrest and initial lodging of breast cancer cells to transmigrate through endothelial cells and extravasate to lodge on to the skeleton in response to its ligand, Fractalkine (FKN/CX3CL1) 49,50 . Targeting CX3CR1 delays the egress of circulating tumor cells from the blood circulation, induces cancer cell apoptosis, and reduces metastasis 51 . DEG analysis shows CX3CR1 is significantly downregulated in Black versus www.nature.com/scientificreports/ White patient tumors (Table 1). CX3CR1 was lower in Asian patient tumor samples based on RNA gene counts (Fig. 2). Molecular subtype analysis showed CX3CR1 is significantly lower in luminal B, HER2 enriched, and triple negative breast tumors than in luminal A (Fig. 3). CX3CR1 expression associated with better overall survival (Fig. 8C) and worse patient survival for luminal A patients (Fig. 10D).

Discussion
We verified that expression differences observed across racial category were not confounded by molecular subtype prevalence in the different racial groups, as it is well established that minorities, such as Black women present more often with triple negative breast cancer and Asian and Latinas with HER2 enriched breast cancers than White women 8,28,29 . The expression of distinct chemokine receptors in the bulk breast cancer tissue could account for a differential recruitment of inflammatory factors such as lymphocyte infiltrate, stromal cells, and cytokine concentration gradients in the tumor microenvironment. The differential expression of these chemokine receptors across racial group category with the same molecular subtype may explain the variability in tumor microenvironment across racial groups and their potential response to future immune therapies. In addition, our analysis highlights the importance of stratifying patient racial differences within tumor molecular subtypes to better understand tumor microenvironment features. Differential gene expression showed that while there are many genes that are differentially expressed, only a few chemokine receptor genes (Table 1) were significantly different when comparing samples from White versus Black or Asian breast cancer patients. Our results warrant further studies of gene expression assessment in breast tumors across diverse patient samples to harness targeting these receptors and their distribution within cell subsets as potential therapeutics to reduce cancer disparities. A major limitation of this study is that we did not access DNA sequencing data to correlate ancestry to the patient samples' race category, and large variations could result from incorrect or mixed self-reported racial group classification. Other limitations include the limited number of tumor samples and metastatic samples within this www.nature.com/scientificreports/ breast cancer cohort, as we could not associate metastatic status to expression level of our genes of interest. In addition, low sample size for minority racial groups in the TCGA makes the survival analysis by race stratification unreliable. While the TCGA is not a population-based study sample, it provides access to hundreds of patient samples with tumor genomics and clinical features making it an important dataset to form the basis of improved tumor targeted therapies, including normal tissue adjacent to the tumor samples 31 . Despite these limitations, the chemokine receptor genes we described have been consistently identified as being involved in metastatic processes and the epithelial to mesenchymal transition, as mentioned above. In addition, here we used bulk RNA-seq findings to hypothesize about the tumor microenvironment's chemokine receptor composition of tumor samples across race, when resolution at the single cell level will be necessary to allow us to elucidate the role of these receptor's cellular distribution across different patient's microenvironments.

Methods
Data collection. We downloaded Breast Invasive Carcinoma RNA-seq count and clinical data from The Cancer Genome Atlas (TCGA) database from 623 White, 58 Black, 52 Asian, 27 Latina and 1 American and Indian/Alaska Native patients, that had a primary tumor sample via the Genomic Data Commons (GDC) Portal on or before July 2020. Level 3 RNA-Seq data was used for this study, which is de-identified and publicly available through TCGA. The study was carried out in accordance with relevant guidelines and regulations.
Weighted gene co-expression network analysis (WGCNA). WGCNA was applied to the normalized, filtered, final expression matrix to identify gene groups (modules). Modules are clusters of highly interconnected genes. Only the top 33% of genes with appreciable expression levels (FPKM > 1) in more than half of the breast cancer patients were subjected to analysis. We proceeded to cut out 14 sample outliers, which resulted in 810 tumor samples for final analysis. We used the WGCNA R package 52,53 with soft-thresholding power 6, to build a weighted gene co-expression network that contained 20,564 nodes (genes) analyzed in one block.
Module-trait relationships. The identified modules were correlated with the available clinical traits available in the TCGA, and also the tumor molecular subtypes and the PAM50 RNA status for the available samples from the TCGA study 30 . Significant modules were plotted against clinical traits. Each row corresponds to a module eigengene, and each column corresponds to a clinical parameter, i.e. sample type, ethnicity, gender, molecular subtype, and PAM50 RNA. The module eigengene is defined as the first principal component of a given module and considered a representative of the gene expression profiles in a module. Each grid contains the correlation value, calculated based on eigengene expression and clinical traits. Listed in the heatmap are bicor correlation rho values and p-values for the correlation (in parentheses), defining relationships between overall weighted expression profiles of modules across samples and clinical traits. The module colors are shown on the left side of each row. The modules and number of genes each module contains are identified by a color, except for the "grey" module, which is reserved for unassigned genes with transcripts with lower correlations across samples not considered strongly co-expressed 52,53 . Differential expression analysis. We used DESeq2, an R package that utilizes negative binomial generalized linear models (GLM) to test for differential expression 54,55 . We analyzed the breast cancer gene expression data from the TCGA to identify genes differentially expressed in Black and Asian patients compared to White patients. We used sample expression and the associated patient metadata. The GLM used to model the effect of cancer diagnosis, stage, race, as well as other covariates such as age, on gene expression levels was: radiation_ therapy + ethnicity + race. Genes differentially expressed due to race were identified by dropping the race factor from the model. Genes with false discovery rate (FDR)-corrected p value < 0.05 were considered significant. Common genes for normal adjacent versus tumor gene lists were identified using Venny 56 .
Gene expression based on clinical parameters. We used RNA-seq HTSeq-counts and clinical sample data from the TCGA breast cancer patients, accessed through the GDC portal. Box and whisker plots represent the interquartile range and median line with a Log10 axis scale showing RNA-seq HTSeq-counts with global significance for ANOVA with Kruskal Wallis.
Survival analysis. For analysis of TCGA breast cancer patients from different racial categories, we used